Telomerase promotes formation of a telomere protective complex in cancer cells.

Telomerase is a ribonucleoprotein complex that catalyzes addition of telomeric DNA repeats to maintain telomeres in replicating cells. Here, we demonstrate that the telomerase protein hTERT performs an additional role at telomeres that is independent of telomerase catalytic activity yet essential for telomere integrity and cell proliferation. Short-term depletion of endogenous hTERT reduced the levels of heat shock protein 70 (Hsp70-1) and the telomere protective protein Apollo at telomeres, and induced telomere deprotection and cell cycle arrest, in the absence of telomere shortening. Short-term expression of hTERT promoted colocalization of Hsp70-1 with telomeres and Apollo and reduced numbers of deprotected telomeres, in a manner independent of telomerase catalytic activity. These data reveal a previously unidentified noncanonical function of hTERT that promotes formation of a telomere protective complex containing Hsp70-1 and Apollo and is essential for sustained proliferation of telomerase-positive cancer cells, likely contributing to the known cancer-promoting effects of both hTERT and Hsp70-1.

The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication.

BACKGROUND: DNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to also possess primase activity, as has been shown in plants; however this activity appears to have been lost in metazoans. Given this, the study of Twinkle in other organisms is required to better understand the evolution of this family and the roles it performs within mitochondria. RESULTS: Here we describe the characterization of a Twinkle homologue, Twm1, in the amoeba Dictyostelium discoideum, a model organism for mitochondrial genetics and disease. We show that Twm1 is important for mitochondrial function as it maintains mitochondrial DNA copy number in vivo. Twm1 is a helicase which unwinds DNA resembling open forks, although it can act upon substrates with a single 3' overhang, albeit less efficiently. Furthermore, unlike human Twinkle, Twm1 has primase activity in vitro. Finally, using a novel in bacterio approach, we demonstrated that Twm1 promotes DNA replication. CONCLUSIONS: We conclude that Twm1 is a replicative mitochondrial DNA helicase which is capable of priming DNA for replication. Our results also suggest that non-metazoan Twinkle could function in the initiation of mitochondrial DNA replication. While further work is required, this study has illuminated several alternative processes of mitochondrial DNA maintenance which might also be performed by the Twinkle family of helicases.

Identification of Pif1 helicases with novel accessory domains in various amoebae.

Pif1 helicases are a conserved family of eukaryotic proteins involved in the maintenance of both nuclear and mitochondrial DNA. These enzymes possess a number of known and putative functions, which facilitate overall genome integrity. Here we have identified multiple subtypes of Pif1 proteins in various pathogenic and non-pathogenic amoeboid species which possess additional domains not present in other Pif1 helicases. These helicases each possess one of five different accessory domains, which have roles in ubiquitination, origin of DNA replication recognition or single-stranded nucleic acid binding activity. Using a robust phylogenetic approach we examined each Pif1 class, which revealed that gene duplication, fusion and horizontal gene transfer events have all contributed to the evolution of these enzymes. This study has identified the first collection of Pif1 helicases to contain additional domains, which likely confer novel enzymatic activity, or improve existing functionality. Furthermore, the potential functions of these helicases may shed further light on the overall role the Pif1 family plays in genome maintenance.

Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption.

BACKGROUND: The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. RESULTS: We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. CONCLUSIONS: Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain.